18 January 2010

Insulin has a number of down-stream targets (receptors), one of which has the highly descriptive name foxa2 (aka hepatocyte nuclear factor 3-beta). This receptor is closely tied to the liver and how it reacts to insulin (Wolfrum, 2004). Essentially foxa2 is one of the regulators responsible for the production of enzymes that are involved in beta-oxidation of fatty acids and the production of ketones. Insulin binds to foxa2, making it unable to perform its function in activating the production of these important fatty-acid metabolism enzymes. In this manner, insulin shuts down fatty acid metabolism.

First a little background on gene expression if you didn't implicitly understand what I wrote above. About 1 % or so of your DNA encodes for proteins. To make a protein, the DNA has to be pulled apart and a complementary single-stranded RNA polymer built to match. This is called messenger RNA and it is sent off to an organelle known as a ribosome which actually assembles the protein that the DNA encodes for.

The rest of your DNA is functional with 'dead space' being a common function. DNA, when being unzipped typically folds in on itself, so sections of the DNA well ahead of the protein encoding region often have functions related whether or not that protein encoding region is actively being transcribed or not. Other proteins literally sit on these spaces and their interaction with the enzymes that unzip and transcribe DNA determine whether messenger RNA is produced or not. Foxa2 is one of these gene transcription activators, so it operates at a very basic level of cellular mechanics. However, it can only do this when insulin is bound to foxa2. Presumably the binding of insulin to foxa2 reconfigures the shape of the foxa2 protein; with proteins, function follows structure/shape.

Silva et al. (2009) published in the journal Nature that this same receptor, foxa2, is found in the hypothalamus (of mice) and it directly effects the hunger reflex. What they did was to take normal and genetically obese mice, fast them, and inject some of them with insulin to put them into the 'fed' state. They then sacrificed the mice and dissected their brains, using an antibody-based stain to identify neurons that were positive for foxa2 and orexin and melanin-concentrating hormone (MCH). Orexin and MCH are known to be associated with feeding and, incidentally, sleep behaviour (Willie, 2001). From their results the authors concluded that the production of the neuropeptides orexin and melanin-concentrating hormone (MCH) were promoted by the foxa2 receptor (but only with insulin attached to it). One of the stronger pieces of evidence was that foxa2 was found in the cytoplasm of the mouse neurons when in the fasted state but in the nucleus when in the fed state. Transcription of messenger RNA occurs in the nucleus.

Supplementary Figure 1 (from Silva, 2009): Author's impression on how the foxa2 receptor cycles in and out of the nucleus in response to insulin.

I put mice in parenthesis in the preceding paragraph because there have been significant differences found between how various pieces of molecular machinery are distributed in rodents versus humans. A good example of this is selano-deiondinase type 2 (D2), which converts the inactive form of thyroid hormone, T4, into the active form T3. It's found in the skeletal muscle of humans but not rats ( Heemstra, 2009 and Larsen, 2009). Incidentally, both papers have fascinating implications for fasting in humans as well as sick euthyroid syndrome.

Interestingly the way they found the distribution of the Foxa2 receptor amongst neurons of the hypothalamus was through the use of specific antibodies as a microscopy fluorescence stain. I'm not clear on how these antibodies are fabricated from the supplemental literature, and a search for, "foxa antibod*" on PubMed didn't return any pertinent hits. I'm sure this is a common method as I've seen it before in fluorescence microscopy, but I am curious about the potential for an associated autoimmune disease.

Interestingly, the Nes-Cre/+;Foxa2T156Aflox/flox allele was associated with dramatic increases in spontaneous locomotor activity relative to control mice (Fig. 3i). The difference between the locomotor activity of Nes-Cre/+;Foxa2T156Aflox/flox mice and that of Foxa2T156Aflox/flox or Nes-Cre/+ mice was similar to the increase in movement of fasted wild-type mice relative to fed wild-type mice (Fig. 3j). The types of physical activity induced inNes-Cre/+;Foxa2T156Aflox/flox mice included searching as well as intense grooming, rearing and face-washing behaviour.

If you can get past the ridiculous names of the mice variants, the English here is pretty clear.

Now it's very easy to get lost in minutiae such as this and lose clarity in the process. Of course, minutiae does have value for the task of bamboozlement. If we pull back and look at the big picture, the key point here is that insulin has been directly implicated in the hunger reflex for the first time, to my knowledge. Previously I assumed that only leptin and ghrelin can effect hunger. Now, when I read this article I did ask the question, has insulin been implicated to interact with this receptor at a biochemical level, or perhaps it is stimulating some other intermediate hormone which in turn interacts with foxa2? The answer is, yes, insulin is the actor and it directly binds to foxa2 (Wolfrum, 2003).

12 January 2010

I have, off and on, entered into discussions with other bloggers on the role of leptin in long-term energy storage. Leptin, we know, is strongly related to long-term storage of fat and is probably one of the primary hormones associated with obesity (Kelesidis, 2006). It is thought, along with ghrelin, to be one of the hormones responsible for appetite.

One question I've posed is does leptin have an antagonist hormone? Most hormones have complements that act to oppose their action. For example, insulin versus glucagon/growth hormone. As an aside, please recall that growth hormone is primarily a catabolic hormone that turns on the body's fat metabolism, a state we call fasted. Generally an antagonist allows the endocrine system to respond more rapidly than simply waiting for the pertinent hormone's concentration in the blood to clear. Does leptin need an antagonist? Or does it operate over such a long time-span that it normally wouldn't need one? Is the lack of an (apparent) antagonist perhaps one of the reasons leptin metabolism can go screwy?

As an alternative to looking into why leptin makes people fat, I thought it might be interesting to examine how a lack of leptin makes people skinny, or anorexic. Anorexia just means 'skinny' refers to a lack of appetite in medical parlance, while anorexia nervosa (AN) refers specifically to the eating disorder that we've all heard about in the news. People can have abnormally low-body fat without having an eating disorder. For example, individuals with cortisol insufficiency (such as Addison's disease, an autoimmune condition involving destruction of the adrenal cortex) tend to have very low body fat levels, but not necessarily a lack of lean body mass. The lack of cortisol just mutes the body's stress response to store an emergency reserve of fat.

One of the markers that characterizes anorexia nervosa is low circulating leptin levels.

Now, leptin likes to interface with the hypothalamus, which is the part of your brain that essentially acts as an interface between the digital-fast (neural) and analogue-slow (endocrine) control systems of the human body. Lot's of things like to interface with the hypothalamus though, so please do not take this role of leptin as dogma. Together, the hypothalamus and pituitary are the master endocrine organ system, regulating the serum concentration of most of the hormones in your body. Essentially it integrates many different signals, and based on those signals decides what quantity of eight primary hormones to release (i.e. oxytocin, argigine vassopressin, adrenocorticotropic hormone, growth-hormone, thyrotropin (TSH), prolactin, luteinizing hormone, and follicle-stimulating hormone). The hypothalamus plays a crucial role in regulating immune function, metabolism, sex function, and mood/anxiety amongst many others.

The hypothalamus (and the pituitary by extension) tends to release hormones in pulses. When I say the hypothalamus exists on the border between digital and analogue that is nearly literally true. The hypothalamus samples the blood-stream for various feedback mechanisms (i.e. hormones) and when it adds together enough signals that indicate the system needs more growth hormone, it generates a pulse. This is done by the combination of neural and endocrine tissues. Leptin is one of the signals that contributes to whether or not pulses are released from the hypothalamus. If everyone's leptin receptor cells are identical, which is not likely, then low leptin levels will probably down-regulate some of the hypothalamic-pituitary hormones and up-regulate some others, while high leptin levels will do the opposite.

One very common side-effect of AN is the loss of the menstrual cycle (which has the scientific name amenorrhea) The menstrual cycle is initiated by a luteinizing hormone pulse, which implies that very low leptin levels have effects beyond simply regulating fat levels. This is not a surprising result; we would expect the body to shut down non-essential functions when it is starving. This result is correlated to circulating leptin levels (Blüher, 2007). Blüher has some interesting comments on the matter of leptin release:

Another side-effect of AN is increased activity (aka hyperactivity), which is a homeostatic method to increase caloric expenditure. This is called activity-based anorexia (ABA) and is one of the primary animal models of anorexia. A review by Hillebrand et al. (2008) shows that leptin itself appears to be signaling the hypothalamus to encourage the brain to engage in this sort of behaviour, and that leptin-replacement therapy suppressed this activity. It's been hypothesized that hyperactivity would promote foraging behaviour in the paleolithic-era and in wild animals. Leptin also has a role in the homeostatic mechanisms behind thermogenesis via the basal metabolism of the thyroid hormones and brown adipose tissue (Rogers, 2009).

This result begs the question, are obese individuals sedentary because they have high circulating leptin levels? Was Gary Taubes, of Good Calories, Bad Calories fame, right in the lack of a relationship between exercise and obesity, even if he didn't know why? If so, hyper/hypoactivity as it relates to leptin would appear to be a case of positive feedback, where the signal tends to reinforce itself over time. It's only because gathering food requires so little energy investment today (get off couch, walk to pantry, grab chips) that this positive feedback cycle blows up so spectacularly. Historically putting on some fat might discourage activity via leptin, giving the organism a rest period.

Now on another front, anorexia nervosa patients who recover from the condition and regain body weight often regain too much and become overweight. This occurred even when caloric-intake and leptin levels were monitored during the body weight gain period to prevent excessive weight gain (Lob, 2003). So once again we see the dominance of the endocrine system and homeostasis over counting calories.

What might cause this higher than normal set-point of body mass index (BMI)? This question does not seem to have a firm answer quite yet so I'm going to speculate. The hypothalamus is a union of neural and endocrine tissue. Neurons, in particular, are quite plastic in that the amount of stimulus you have to apply to get them to fire changes depending on their exposure history. This is how memory is thought to work, for example. My hypothesis is that the neural component of the hypothalamus habituates to long-term leptin exposure.

There are clearly some threshold levels where leptin indicates an organism is in semi-starvation mode and generates compensatory behaviour (Müller, 2009). I can postulate that there may also be hibernation morphology at the top-end of the leptin spectrum. If the organism stays in semi-starvation mode for long enough, perhaps the sensitivity to leptin in the hypothalamus is reduced by the plasticity of the neural component. In this case, a crash weight-gain diet would not give the hypothalamus's neurons sufficient time to change their sensitivity to leptin, and adapt a new set point.

Maybe this is the reason why fast weight-loss programs typically fail miserably. The leptin set points for semi-starvation modes are at at abnormal levels, and pushing leptin through them induces behaviour that likely results in a rebound. The solution then is to be patient and go slow with weight loss or gain. If my hypothesis is correct, losing weight too fast may actually permanently distort leptin regulation.